formulation and evaluation of liposomal gel for
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Int J Pharm Bio Sci 2013 Oct; 4(4): (P) 22 – 32
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'esearch rticle o*el r+, eli*er- s-stem
International Jo+rnal o" Pharma an Bio Sciences ISS
0./&2..
O'5TIO 6 785TIO O 5IPOSO5 975 O'
T'7T7T O PSO'ISIS.
D. G. UMALKAR * AND RAJESH K.S
Parul Institute of Pharmacy,Limda,Vadodara , India.
ABSTRACT
The aim of this study was to formulate and evaluate liposomal gel of Diflurasonediacetate for topical application. Liposomes were prepared by using Soya PC and EggPC by the lipid film hydration method. It was characteried by DSC and Surfacemorphology of Liposomes by SE!. assessed for particle sie" entrapment efficiency"and their effect on in vitro drug release. The #atch #$ containing Soya PC withCholesterol in %&' was found to be optimied .The Stability study of optimiedformulation was done as per IC( guidelines. The )ptimied liposomes wereincorporated into Carbopol gel *+,- and were evaluated for ex vivo drug permeationstudies. It was observed that Diflurasone diacetate loaded liposomes bearing hydrogelwas more efficient in the treatment of Psoriasis.
KEYWORDS: Diflurasone diacetate; Liposomes; Topical gels; Topical delivery
.
D. G. UMALKAR
Par+l Instit+te o" Pharmac-5ima8aoara Inia#
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Int J Pharm Bio Sci 2013 Oct; 4(4): (P) 22 – 32
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INTRODUCTION
During the past several years" Liposomes beganto act as a topical carrier for pharmaceuticalmolecules. Liposomes have emerged as analternative to conventional carriers such ascream" tincture and emulsion'"+. Liposomescombine their advantages such as controlledrelease" in vivo good toleration and protection ofactive compounds. Liposomes can favor drugpenetration into the s/ins$" 0" maintain asustained release to avoid systemic absorption1"act as a depot system2 and reduce irritation3" %.Diflurasone diacetate is a topical Corticosteroidsacts by the induction of phospholipase4+ inhibitory proteins" collectively calledlipocortins. It is postulated that these proteinscontrol the biosynthesis of potent mediators ofinflammation such as prostaglandins andleu/otrienes by inhibiting the release of theircommon precursor arachidonic acid. 4rachidonicacid is released from membrane phospholipidsby phospholipase 4+. The drug is primarily usedas a topical treatment for Psoriasis'5" ''. Thetreatment for psoriasis includes topical therapy"systemic therapy and phototherapy. Thesystemic therapy leads to systemic to6icity aspsoriasis re7uires long term therapy and
phototherapy is e6pensive as well as leads topoor patient compliance. Topical corticosteroidformulations are available in mar/et inconventional form having disadvantage of lowtransdermal penetration which leads to lowtherapeutic effectiveness. The rationale behindthis wor/ was to develop liposomal Topical Drug
Delivery System with enhance permeation and tosustain the drug release at the localied areaThe aim of this study was to develop topical gelscontaining liposomal dispersions loaded withDiflurasone diacetate.
MATERIALS AND METHODS
Diflurasone diacetate was gifted by !icro Lab#angalore" India. Phospholipid *Soya PC- wasobtained from Lipoid 8mb(" 8ermanyCholesterol" Carbopol %$P were purchasedfrom SD 9ine Chemicals" !umbai" India. 4ll theother chemicals were of the analytical grade
:ater was used in double;distilled 7uality.
Preparation of Liposomal Dispersion 12
The liposomal dispersions were prepared usingLipid film (ydration method. Table' reports thecomposition of the prepared liposomadispersions. Drug" Soya PC<Egg PCCholesterol was dissolved in Chloroform!ethanol *&' v<v- = solvent was evaporated in>ota evaporator *E7itron >oteva- under reducedpressure at 15 o c = 15 rpm for '0 min until a dry
film was formed. 9ilm was hydrated with '5mPhosphate buffer p(1.3 = rotated for '5 min. toget uniform dispersion. It was sonicated at $ o cusing probe sonicator *at $5 w" vibra cell" modeno.C? $" S)@ICS- in cycles of 0 minproviding 0 min rest between each cycle.
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Table IComposition of various Liposomal formulations. Particle size,
Polydispersity Index (PI), eta potential and ! Dru" entrapment,!Dru" #elease parameters of different Liposomal formulations
C$aracterization of Liposomal DispersionParticle size and zeta potential
determination
2%
Particle sie and sie distributionmeasurements of the liposomal dispersionswere performed using photon correlationspectroscopy *PCS-. The average particle sie*;average sie- and polydispersity inde6 *PDI-were measured by photon correlationspectroscopy !alvern Aetasier at +0 o C undera fi6ed angle of %5o in disposable polystyrenecuvettes. Aeta potential was measured by usingAetasier. Samples were placed in clear eta
cells and results were recorded. #efore puttingthe fresh sample" cuvettes were washed with
the methanol and rinsed using the sample to bemeasured before each e6periment.
!Dru" entrapment efficiency &'
The amount of encapsulated Diflurasonediacetate was calculated by subtracting the freeamount of the drug from Diflurasone diacetateliposomal dispersion by ultracentrifugation at+5"555 rpm at '5 o C for 5 min. 4ftecentrifugation" ' ml of supernatant was dilutedwith the addition of % ml phosphate saline buffer*p( 2.$- and then the absorbance wasmeasured using B??is spectrophotometer at
+3 nm..Entrapment efficiency *EE ,- wascalculated from the following e7uation
4mount of drug actually present
EE '55Theoretical drug laded e6pected
cannin" electron microscopy (*) &1 The morphology *shape and surface
characteristics- of Liposomes was studied byscanning electron microscopy *SE!- *modelFS!;01'5L? Scanning !icroscopeG Feol" To/yo"Fapan-. The sputtering was done for nearly 0minutes to obtain uniform coating on the sampleto enable good 7uality SE! images. The SE!was operated at low accelerating voltage ofabout '0H? with load current of about 35!4.The condenser lens position was maintainedbetween $.$ to 0.'. The obective lens aperture
has a diameter of +$5 microns and the wor/ingdistance :D%mm.
DC +nalysis &1 DSC studies of pure Diflurasone diacetatecholesterol" soya pc = mi6ture was carried out.4ccurately weighed samples were carefullyplaced in DSC boats and heating curves wererecorded in temperature range of 25–250 ◦C at aheating rate of 10 ◦C/min under inert atmosphere*N 2 30 ml/min-.The study was carried out usingDifferential Scanning Calorimeter *DSC" !ettler
Batch
code
Drug : Soya
PC ratio
Drug : gg
PC ratio
PC:Cholest
erol ratio
Particle si!e
"nm#
PDI $eta potential
"m%#
Drug entrapment
"&#
Drug 'ele
"&#B( (:( ; ):( +55.%5J+.'5 5.31 ;$5. J 5.2 20.+ J5.%+ 2+.'1J5.3B* (:* ; ):( +'5.%5J'.+ 5.+%% ;$$.$ J +.02 21.10 J5.3' 10.+$J5.%B+ (:+ ; ):( +'3.55J'.10 5.13$ ;05.+ J .' 3+.15 J5.%5 13.5%J5.0B, (:, ; ):( +3.51J'.$5 5.1+ ;$5.0+ J 5.32 3$.+3 J5.0 35.51J5.0B- (:- ; ):( '+.05J'.+$ 5.10+ ;$%.+ J 5.20 23.+$ J5.1 2$.53J5.0B. ; (:( ):( +'+.%J'.12 5.35$ ;05.2 J 5.32 2$.03 J5.02 2'.+J5.13B/ ; (:* ):( +2$.2J+.'5 5.1+0 ;+.0 J '.12 20.0$ J5.33 12.03J5.%B0 ; (:+ ):( +30.25J'.05 5.03% ;. J '.3$ 35.+0 J5.02 12.11J5.$B) ; (:, ):( 01.+5J'.1 5.2' ;0.3 J '.03 3'.5 J5.1$ 1$.%J5.3B(1 ; (:- ):( $33.$5J'.15 5.31 5.+'J'.+0 22.%$ J5.$3 0%.$$J5.0
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Toledo Star e"S: 2.5' Fapan-.
In itro Dru" #elease &2 The in vitro drug release profile of Diflurasonediacetate loaded Liposome;bearing hydrogel andmar/eted formulation were studied using a
dialysis bag. 9ormulations were ta/en into adialysis bag *molecular weight cut;off" 3555- andplaced in a bea/er containing +5 ml of P#S *p(1.3- Then" the bea/er was placed over amagnetic stirrer and the temperature of theassembly was maintained at 2 J 'KCthroughout the study. Samples *+ ml- werewithdrawn at definite time intervals and replacedwith e7ual amounts of fresh buffer. The sampleswere analyed for drug concentration by B?;?ISspectrophotometer at +% nm.
ta-ility tudies&2 In order to determine the physical stability ofvesicles" the )ptimied vesicles *#atch # $- wasstored at $ o c and $5 o c for up to monthsunder light protection. In predetermined timeintervals" the particle sie and EntrapmentEfficiency of the vesicles was measured
Preparation and C$aracterization ofLiposomal/ased 0ydro"el &2
9or the preparation of hydrogel" the gel;formingpolymer Carbopol %$P*+,- was dispersed indistilled water" stirred for '5 min at '055 rpm andneutralied by triethanolamine until p( 1.5.(ydrogel were further allowed to e7uilibrate for+$ hours at room temperature and then used todisperse a freshly prepared Liposomalsuspension. Liposomal dispersion and hydrogelwere mi6ed in a high speed stirrer *>emi"!umbai" India- at '555 rpm for the ne6t 0 min.The gel was allowed to stand overnight to
remove entrapped air.
x vivo sin permeation tudies 21
E6 vivo permeation of Diflurasone diacetate fromLiposome based gel and mar/eted formulation*Diflorate cream - were performed using themodified 9ran Diffusion Cell. The animals weresacrificed by an overdose of chloroforminhalation. The hairs on the dorsal side of animalwere removed with the help of 5.' mm animal
hair clipper" in the direction of tail to head. Theshaven part of the s/in was separated from theanimal and the hypodermis including bloodvessels were surgically removed using a surgicablade. The dermis part of the s/in was wiped offwith a wet cotton swab soa/ed in iso propanol
$ times to remove any adhering fat material. Thes/in membrane surface area e6posed toreceptor phase was 0.2++1 cm+ the preparedformulations each e7uivalent to '.5 mg of drugwas applied onto the prepared mice s/in facingthe donor chamber. 4n ali7uot of 0 ml ofsamples was withdrawn at suitable time intervaand replaced with same amount of medium tomaintain the receptor phase volume as $0 mlThe samples were analysed by B?spectrophotometer at +3 nm.
ittin" into 3inetic *odelThe diffusion data obtained from diffusion profilewas fitted by to various /inetic models *Aeroorder" 9irst order" (iguchi" Horesmeyer peppas-and the best fit model was obtained byregression analysis. The release mechanism isdetermined by finding out n value form peppasmodel.
RESULTS & DISCUSSION
Particle size and eta potentiadetermination The average particle sie analyed by !alvernParticle sie 4nalyer was found to be between+55.%5J+.'5 to $33.$5J'.15 nm which is in thedesired range as shown in table @o.'. The Polydispersity inde6 *PDI-" is a measure ofdistribution of molecular mass in a given sampleThe PDI calculated is the weight averagemolecular weight divided by number averagemolecular weight. The PDI has value less than5.0" this indicates good dispersion. It shows thatas the concentration of lipid increases theparticle sie also increases .Aeta potential is anindication of the stability of the vesicle andindicates charge present on the vesicularsystem. (ere eta Potential was found to be ;;$5.0+ J 5.32 m? for #$ formulation whichindicates a negative surface charge on
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Liposomes this shows stability of Preparationbecause of the anticipated surface repulsionbetween similar charged particles henceinhibiting aggregation of Liposomal vesicularparticle.
4 !Dru" entrapment efficiency#atch #$ shows highest , Entrapment
efficiency of 3$.+3J5.0. 9rom the data obtainedfor entrapment efficiency we can say that as theconcentration of lipid increases the entrapmentof the drug also increases for lipophillic drug as
the nature of the lipid is also lipophillicCholesterol also plays an important role inentrapment efficiency. (ere as the concentrationof cholesterol increases the entrapmenincreases upto certain e6tent but further increasein cholesterol causes decrease in entrapment
cannin" electron microscopy 5 *SE! Photographs indicated that the preparedLiposomes were nearly spherical in shape asshown in fig.@o.'
2igure 3o (* of Liposomes
• DC +nalysisDSC studies of pure Diflurasone diacetate" cholesterol" soya pc = mi6ture was carried out and resultswere given in fig. @o.+ and fig.@o.. 9rom the thermograms" it was concluded that drug and e6cipientare compatible with each other.
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2igure 3o * DC Diflurasone diacetate
2igure 3o + DSC4 Liposomes
• In itro Dru" #elease
The graph of drug diffusion shows that drug release of batch #$ containing Soya PC was found to bema6imum release of 35.51J5.0 after +$ hrs.
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ummaryThe Liposomes was prepared by the thin film
hydration method. The optimied formulations#atch #$ showed the Particle sie of+3.51J'.$5 = PDI of 5.1+ with acceptableentrapment efficiency of 3$.+3 and drug releaseof 35.51 after +$ hrs. SE! Photographsindicated that the prepared Liposomes werenearly spherical in shape. DSC analysisconcluded that drug and e6cipient arecompatible with each other. Stability studyreveals that )ptimies Liposomal formulation*#atch #$- was found to be stable. The
)ptimied formulation #atch #$ gel wasprepared by using Carbopols%2$P = wasevaluated for Ex vivo s/in permeation studiesand it shows 25.51 , CD> after +$ hours.?arious Hinetic model was use to study the
release pattern and it shows Aero order releasewith anomalous diffusion.
CONCLUSION
The Diflurasone diacetate loaded Liposomescould be fabricated with the help of a thin filmhydration method and successfully incorporatedinto hydrogel for topical application. The in vitroand e6 vivo data indicate that Diflurasonediacetate loaded Liposomes bearing hydrogeprovides an e6cellent sustained release ofDiflurasone diacetate. The obtained results
reflect the potential of Liposomes as a carrierfor topical administration of Diflurasonediacetate. In conclusion" the developed systemsare promising alternative drug carriers fotopical pharmaceutics.
ACKNOWLEDGMENT
The author ac/nowledges !icro labs Pvt. Ltd." #angalore *India- for providing Drug sample" Lipoid8ermany for providing lipids and Parul Institute of Pharmacy and F@TB" (yderabad for providingresearch facility and encouragement for this research wor/.
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